A mutation-independent approach via transcriptional upregulation of a disease modifier gene rescues muscular dystrophy in vivo

Abstract

Introductory paragraphIdentification of protective and/or pathogenic genetic modifiers provides important insight into the heterogeneity of disease presentations in individuals affected by neuromuscular disorders (NMDs), despite having well-defined pathogenic variants. Targeting modifier genes to improve disease phenotypes could be especially beneficial in cases where the causative genes are large, structurally complex and the mutations are heterogeneous. Here, we report a mutation-independent strategy to upregulate expression of a compensatory disease-modifying gene in Congenital Muscular Dystrophy type 1A (MDC1A) using a CRISPR/dCas9-based transcriptional activation system. MDC1A is caused by nonfunctional Laminin α2, which compromises muscle fibers stability and axon myelination in peripheral nerves 1. Transgenic overexpression of Lama1, encoding a structurally similar protein Laminin α1, ameliorates muscle wasting and paralysis in the MDC1A mouse models, demonstrating its role as a protective disease modifier 2. Yet, upregulation of Lama1 as a postnatal gene therapy is hampered by its large size, which exceeds the current genome packaging capacity of clinically relevant delivery vehicles such as adeno-associated viral vectors (AAVs). In this study, we sought to upregulate Lama1 using CRISPR/dCas9-based transcriptional activation system, comprised of catalytically inactive S. aureus Cas9 (dCas9) fused to VP64 transactivation domains and sgRNAs targeting the Lama1 promoter. We first demonstrated robust upregulation of Lama1 in myoblasts, and following AAV9-mediated intramuscular delivery, in skeletal muscles of dy2j/dy2j mouse model of MDC1A.We therefore assessed whether systemic upregulation of Lama1 would yield therapeutic benefits in dy2j/dy2j mice. When the intervention was started early in pre-symptomatic dy2j/dy2j mice, Lama1 upregulation prevented muscle fibrosis and hindlimb paralysis. An important question for future therapeutic approaches for a variety of disorders concerns the therapeutic window and phenotypic reversibility. This is particularly true for muscular dystrophies as it has long been hypothesized that fibrotic changes in skeletal muscle represent an irreversible disease state that would impair any therapeutic intervention at advanced stages of the disease. Here, we demonstrate that dystrophic features and disease progression were significantly improved and partially reversed when the treatment was initiated in symptomatic 3-week old dy2j/dy2j mice with already-apparent hind limb paralysis and significant muscle fibrosis.Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR/dCas9-mediated modulation of a disease modifier gene, which opens up an entirely new and mutation-independent treatment approach for all MDC1A patients. Moreover, this treatment strategy provides evidence that muscle fibrosis can be reversible, thus extending the therapeutic window for this disorder. Our data provide a proof-of-concept strategy that can be applied to a variety of disease modifier genes and a powerful therapeutic approach for various inherited and acquired diseases.

Development of therapeutic genome engineering in laminin-α2-deficient congenital muscular dystrophy

“…This construct was guided to the Lama1 proximal promoter region by a combination of three sgRNAs. We showed that systemic administration of AAV9-carrying the CRISPR/dCas9 components led to robust up-regulation of laminin-α1 in the dy 2j /dy 2j mice, resulting in the amelioration of dystrophic features in skeletal muscle and improvement in mobility and muscle functions of the mice [25].…”

“…Concerns over the long-term safety of delivering genome-modifying cargo to cells and host immune responses to the AAV vectors and the cargo must also be addressed. In the case of MDC1A, amelioration of disease phenotypes in vivo highly depends on efficient targeting in both the skeletal muscles and peripheral nerves [21,25]. Improvements of the biodistribution, tissue penetration and, in particular, editing efficiency are required in order to fully exploit the potential of therapeutic AAV delivery.…”

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond

“…Additionally, dCas9-VP160 was used in rag/mdx mice to upregulate the laminin subunit alpha 1 chain (Lama1) to increase laminin-111 to stabilize the muscle fiber membrane (102). For MDC1A, a non-mutation specific approach with SadCas9-2xVP64 was used to upregulate Lama1, which can compensate to rescue the dy 2J /dy 2J phenotype in vivo (59).…”

“…Using two vectors for one platform may effectively halve the effective dose. SaCas9 is an alternative, smaller nuclease that can be packaged along with a gRNA into a single AAV and has been successfully applied in some models of NMDs (15,36,59,60,97,98,104,110,122,131). However, a higher percentage of the population is estimated to have pre-existing immunity to SaCas9 compared with SpCas9 (23,119).…”

“…Heart function was assessed and demonstrated improvement in some DMD models (50,110). Last, for other diseases, open-field and tetanic force were improved in a mouse model of MDC1A (59,60), and less myotonia was observed in a model of DM1 (104) after CRISPR-based therapies were given. These studies are promising; however, the long-term benefit of gene editing on functional outcomes will still need to be fully explored.…”